The physiological characteristics of the yeast Dekkera bruxellensis in fully fermentative conditions with cell recycling and in mixed cultures with Saccharomyces cerevisiae

The yeast Dekkera bruxellensis plays an important role in industrial fermentation processes, either as a contaminant or as a fermenting yeast. In this study, an analysis has been conducted of the fermentation characteristics of several industrial D. bruxellensis strains collected from distilleries from the Southeast and Northeast of Brazil, compared with Saccharomyces cerevisiae. It was found that all the strains of D. bruxellensis showed a lower fermentative capacity as a result of inefficient sugar assimilation, especially sucrose, under anaerobiosis, which is called the Custer effect. In addition, most of the sugar consumed by D. bruxellensis seemed to be used for biomass production, as was observed by the increase of its cell population during the fermentation recycles. In mixed populations, the surplus of D. bruxellensis over S. cerevisiae population could not be attributed to organic acid production by the first yeast, as previously suggested. Moreover, both yeast species showed similar sensitivity to lactic and acetic acids and were equally resistant to ethanol, when added exogenously to the fermentation medium. Thus, the effects that lead to the employment of D. bruxellensis in an industrial process and its effects on the production of ethanol are multivariate. The difficulty of using this yeast for ethanol production is that it requires the elimination of the Custer effect to allow an increase in the assimilation of sugar under anaerobic conditions.     

The ability to use nitrate confers advantage to <Emphasis Type="Italic">Dekkera bruxellensis</Emphasis> over <Emphasis Type="Italic">S. cerevisiae</Emphasis> and can explain its adaptation to industrial fermentation processes

The yeast Dekkera bruxellensis has been regarded as a contamination problem in industrial ethanol production because it can replace the originally inoculated Saccharomyces cerevisiae strains. The present study deals with the influence of nitrate on the relative competitiveness of D. bruxellensis and S. cerevisiae in sugar cane ethanol fermentations. The industrial strain D. bruxellensis GDB 248 showed higher growth rates than S. cerevisiae JP1 strain in mixed ammonia/nitrate media, and nitrate assimilation genes were only slightly repressed by ammonia. These characteristics rendered D. bruxellensis cells with an ability to overcome S. cerevisiae populations in both synthetic medium and in sugar cane juice. The results were corroborated by data from industrial fermentations that showed a correlation between high nitrate concentrations and high D. bruxellensis cell counts. Moreover, the presence of nitrate increased fermentation efficiency of D. bruxellensis cells in anaerobic conditions, which may explain the maintenance of ethanol production in the presence of D. bruxellensis in industrial processes. The presence of high levels of nitrate in sugar cane juice may be due to its inefficient conversion by plant metabolism in certain soil types and could explain the periodical episodes of D. bruxellensis colonization of Brazilian ethanol plants.     

The fermentation of sugarcane molasses by Dekkera bruxellensis and the mobilization of reserve carbohydrates

The yeast Dekkera bruxellensis is considered to be very well adapted to industrial environments, in Brazil, USA, Canada and European Countries, when different substrates are used in alcoholic fermentations. Our previous study described its fermentative profile with a sugarcane juice substrate. In this study, we have extended its physiological evaluation to fermentation situations by using sugarcane molasses as a substrate to replicate industrial working conditions. The results have confirmed the previous reports of the low capacity of D. bruxellensis cells to assimilate sucrose, which seems to be the main factor that can cause a bottleneck in its use as fermentative yeast. Furthermore, the cells of D. bruxellensis showed a tendency to deviate most of sugar available for biomass and organic acids (lactic and acetic) compared with Saccharomyces cerevisiae, when calculated on the basis of their respective yields. As well as this, the acetate production from molasses medium by both yeasts was in marked contrast with the previous data on sugarcane juice. Glycerol and ethanol production by D. bruxellensis cells achieved levels of 33 and 53 % of the S. cerevisiae, respectively. However, the ethanol yield was similar for both yeasts. It is worth noting that this yeast did not accumulate trehalose when the intracellular glycogen content was 30 % lower than in S. cerevisiae. The lack of trehalose did not affect yeast viability under fermentation conditions. Thus, the adaptive success of D. bruxellensis under industrial fermentation conditions seems to be unrelated to the production of these reserve carbohydrates.     

The consequences of Lactobacillus vini and Dekkera bruxellensis as contaminants of the sugarcane-based ethanol fermentation

This work describes the effects of the presence of the yeast Dekkera bruxellensis and the bacterium Lactobacillus vini on the industrial production of ethanol from sugarcane fermentation. Both contaminants were quantified in industrial samples, and their presence was correlated to a decrease in ethanol concentration and accumulation of sugar. Then, laboratory mixed-cell fermentations were carried out to evaluate the effects of these presumed contaminants on the viability of Saccharomyces cerevisiae and the overall ethanol yield. The results showed that high residual sugar seemed the most significant factor arising from the presence of D. bruxellensis in the industrial process when compared to pure S. cerevisiae cultures. Moreover, when L. vini was added to S. cerevisiae cultures it did not appear to affect the yeast cells by any kind of antagonistic effect under stable fermentations. In addition, when L. vini was added to D. bruxellensis cultures, it showed signs of being able to stimulate the fermentative activity of the yeast cells in a way that led to an increase in the ethanol yield.     

Effects of single and combined cell treatments based on low pH and high concentrations of ethanol on the growth and fermentation of Dekkera bruxellensis and Saccharomyces cerevisiae

The alcoholic fermentation in Brazil displays some peculiarities because the yeast used is recycled in a non-aseptic process. After centrifugation, the cells are treated with acid to control the bacterial growth. However, it is difficult to manage the indigenous yeasts without affecting the main culture of Saccharomyces cerevisiae. This work evaluated how the cell treatment could be modified to combat contaminant yeasts based on the differential sensitivities to low pH and high concentrations of ethanol displayed by an industrial strain of S. cerevisiae and three strains of Dekkera bruxellensis, which are common contaminant yeasts in Brazilian fermentation processes. The tests were initially performed in rich medium with a low pH or a high concentration of ethanol to analyse the yeast growth profile. Then, the single and combined effects of low pH and ethanol concentration on the yeast cell viability were evaluated under non-proliferative conditions. The effects on the fermentation parameters were also verified. S. cerevisiae grew best when not subjected to the stresses, but this yeast and D. bruxellensis had similar growth kinetics when exposed to a low pH or increased ethanol concentrations. However, the combined treatments of low pH (2.0) and ethanol (11 or 13 %) resulted in a decrease of D. bruxellensis cell viability almost three times higher than of S. cerevisiae, which was only slightly affected by all cell treatments. The initial viability of the treated cells was restored within 8 h of growth in sugar cane juice, with the exception of the combined treatment for D. bruxellensis. The ethanol-based cell treatment, in despite of slowing the fermentation, could decrease and maintain D. bruxellensis population under control while S. cerevisiae was taking over the fermentation along six fermentative cycles. These results indicate that it may be possible to control the growth of D. bruxellensis without major effects on S. cerevisiae. The cells could be treated between the fermentation cycles by the parcelled addition of 13 % ethanol to the tanks in which the yeast cream is treated with sulphuric acid at pH 2.0.     

Interaction of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>–<Emphasis Type="Italic">Lactobacillus fermentum</Emphasis>–<Emphasis Type="Italic">Dekkera bruxellensis</Emphasis> and feedstock on fuel ethanol fermentation

The alcoholic fermentation for fuel ethanol production in Brazil occurs in the presence of several microorganisms present with the starter strain of Saccharomyces cerevisiae in sugarcane musts. It is expected that a multitude of microbial interactions may exist and impact on the fermentation yield. The yeast Dekkera bruxellensis and the bacterium Lactobacillus fermentum are important and frequent contaminants of industrial processes, although reports on the effects of both microorganisms simultaneously in ethanolic fermentation are scarce. The aim of this work was to determine the effects and interactions of both contaminants on the ethanolic fermentation carried out by the industrial yeast S. cerevisiae PE-2 in two different feedstocks (sugarcane juice and molasses) by running multiple batch fermentations with the starter yeast in pure or co-cultures with D. bruxellensis and/or L. fermentum. The fermentations contaminated with D. bruxellensis or L. fermentum or both together resulted in a lower average yield of ethanol, but it was higher in molasses than that of sugarcane juice. The decrease in the CFU number of S. cerevisiae was verified only in co-cultures with both D. bruxellensis and L. fermentum concomitant with higher residual sucrose concentration, lower glycerol and organic acid production in spite of a high reduction in the medium pH in both feedstocks. The growth of D. bruxellensis was stimulated in the presence of L. fermentum resulting in a more pronounced effect on the fermentation parameters than the effects of contamination by each microorganism individually.     

Fermentation characteristics of Dekkera bruxellensis strains

The influence of pH, temperature and carbon source (glucose and maltose) on growth rate and ethanol yield of Dekkera bruxellensis was investigated using a full-factorial design. Growth rate and ethanol yield were lower on maltose than on glucose. In controlled oxygen-limited batch cultivations, the ethanol yield of the different combinations varied from 0.42 to 0.45 g (g glucose)⁻¹ and growth rates varied from 0.037 to 0.050 h⁻¹. The effect of temperature on growth rate and ethanol yield was negligible. It was not possible to model neither growth rate nor ethanol yield from the full-factorial design, as only marginal differences were observed in the conditions tested. When comparing three D. bruxellensis strains and two industrial isolates of Saccharomyces cerevisiae, S. cerevisiae grew five times faster, but the ethanol yields were 0–13% lower. The glycerol yields of S. cerevisiae strains were up to six-fold higher compared to D. bruxellensis, and the biomass yields reached only 72–84% of D. bruxellensis. Our results demonstrate that D. bruxellensis is robust to large changes in pH and temperature and may have a more energy-efficient metabolism under oxygen limitation than S. cerevisiae.     

Enhanced production of ethanol from glycerol by engineered Hansenula polymorpha expressing pyruvate decarboxylase and aldehyde dehydrogenase genes from Zymomonas mobilis

To improve production of ethanol from glycerol, the methylotrophic yeast Hansenula polymorpha was engineered to express the pdc and adhB genes encoding pyruvate decarboxylase and aldehyde dehydrogenase II from Zymomonas mobilis, respectively, under the control of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter. The ethanol yield was 3.3-fold higher (2.74 g l^?1) in the engineered yeast compared with the parent strain (0.83 g l^?1). Further engineering to stimulate glycerol utilization in the recombinant strain via expression of dhaD and dhaKLM genes from Klebsiella pneumoniae encoding glycerol dehydrogenase and dehydroxyacetone kinase, respectively, resulted in a 3.7-fold increase (3.1 g l^?1) in ethanol yield.     

Oxygen- and Glucose-Dependent Regulation of Central Carbon Metabolism in Pichia anomala

We investigated the regulation of the central aerobic and hypoxic metabolism of the biocontrol and non-Saccharomyces wine yeast Pichia anomala. In aerobic batch culture, P. anomala grows in the respiratory mode with a high biomass yield (0.59 g [dry weight] of cells g of glucose⁻¹) and marginal ethanol, glycerol, acetate, and ethyl acetate production. Oxygen limitation, but not glucose pulse, induced fermentation with substantial ethanol production and 10-fold-increased ethyl acetate production. Despite low or absent ethanol formation, the activities of pyruvate decarboxylase and alcohol dehydrogenase were high during aerobic growth on glucose or succinate. No activation of these enzyme activities was observed after a glucose pulse. However, after the shift to oxygen limitation, both enzymes were activated threefold. Metabolic flux analysis revealed that the tricarboxylic acid pathway operates as a cycle during aerobic batch culture and as a two-branched pathway under oxygen limitation. Glucose catabolism through the pentose phosphate pathway was lower during oxygen limitation than under aerobic growth. Overall, our results demonstrate that P. anomala exhibits a Pasteur effect and not a Crabtree effect, i.e., oxygen availability, but not glucose concentration, is the main stimulus for the regulation of the central carbon metabolism.     

Real-Time PCR Assay for Detection and Enumeration of Dekkera bruxellensis in Wine

Traditional methods to detect the spoilage yeast Dekkera bruxellensis from wine involve lengthy enrichments. To overcome this difficulty, we developed a quantitative real-time PCR method to directly detect and enumerate D. bruxellensis in wine. Specific PCR primers to D. bruxellensis were designed to the 26S rRNA gene, and nontarget yeast and bacteria common to the winery environment were not amplified. The assay was linear over a range of cell concentrations (6 log units) and could detect as little as 1 cell per ml in wine. The addition of large amounts of nontarget yeasts did not impact the efficiency of the assay. This method will be helpful to identify possible routes of D. bruxellensis infection in winery environments. Moreover, the time involved in performing the assay (3 h) should enable winemakers to more quickly make wine processing decisions in order to reduce the threat of spoilage by D. bruxellensis.     

Utilization of nitrate abolishes the “Custers effect” in Dekkera bruxellensis and determines a different pattern of fermentation products

Nitrate is one of the most abundant nitrogen sources in nature. Several yeast species have been shown to be able to assimilate nitrate and nitrite, but the metabolic pathway has been studied in very few of them. Dekkera bruxellensis can use nitrate as sole nitrogen source and this metabolic characteristic can render D. bruxellensis able to overcome S. cerevisiae populations in industrial bioethanol fermentations. In order to better characterize how nitrate utilization affects carbon metabolism and the yields of the fermentation products, we investigated this trait in defined media under well-controlled aerobic and anaerobic conditions. Our experiments showed that in D. bruxellensis, utilization of nitrate determines a different pattern of fermentation products. Acetic acid, instead of ethanol, became in fact the main product of glucose metabolism under aerobic conditions. We have also demonstrated that under anaerobic conditions, nitrate assimilation abolishes the “Custers effect”, in this way improving its fermentative metabolism. This can offer a new strategy, besides aeration, to sustain growth and ethanol production for the employment of this yeast in industrial processes.     

Fermentation of lignocellulosic hydrolysate by the alternative industrial ethanol yeast Dekkera bruxellensis

Aim: Testing the ability of the alternative ethanol production yeast Dekkera bruxellensis to produce ethanol from lignocellulose hydrolysate and comparing it to Saccharomyces cerevisiae. Methods and Results: Industrial isolates of D. bruxellensis and S. cerevisiae were cultivated in small‐scale batch fermentations of enzymatically hydrolysed steam exploded aspen sawdust. Different dilutions of hydrolysate were tested. None of the yeasts grew in undiluted or 1 : 2 diluted hydrolysate [final glucose concentration always adjusted to 40 g l^?1 (0·22 mol l^?1)]. This was most likely due to the presence of inhibitors such as acetate or furfural. In 1 : 5 hydrolysate, S. cerevisiae grew, but not D. bruxellensis, and in 1 : 10 hydrolysate, both yeasts grew. An external vitamin source (e.g. yeast extract) was essential for growth of D. bruxellensis in this lignocellulosic hydrolysate and strongly stimulated S. cerevisiae growth and ethanol production. Ethanol yields of 0·42 ± 0·01 g ethanol (g glucose)^?1 were observed for both yeasts in 1 : 10 hydrolysate. In small‐scale continuous cultures with cell recirculation, with a gradual increase in the hydrolysate concentration, D. bruxellensis was able to grow in 1 : 5 hydrolysate. In bioreactor experiments with cell recirculation, hydrolysate contents were increased up to 1 : 2 hydrolysate, without significant losses in ethanol yields for both yeasts and only slight differences in viable cell counts, indicating an ability of both yeasts to adapt to toxic compounds in the hydrolysate. Conclusions: Dekkera bruxellensis and S. cerevisiae have a similar potential to ferment lignocellulose hydrolysate to ethanol and to adapt to fermentation inhibitors in the hydrolysate. Significance and Impact of the study: This is the first study investigating the potential of D. bruxellensis to ferment lignocellulosic hydrolysate. Its high competitiveness in industrial fermentations makes D. bruxellensis an interesting alternative for ethanol production from those substrates.     

Elimination of glycerol and replacement with alternative products in ethanol fermentation by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Glycerol is a major by-product of ethanol fermentation by Saccharomyces cerevisiae and typically 2–3% of the sugar fermented is converted to glycerol. Replacing the NAD⁺-regenerating glycerol pathway in S. cerevisiae with alternative NADH reoxidation pathways may be useful to produce metabolites of biotechnological relevance. Under fermentative conditions yeast reoxidizes excess NADH through glycerol production which involves NADH-dependent glycerol-3-phosphate dehydrogenases (Gpd1p and Gpd2p). Deletion of these two genes limits fermentative activity under anaerobic conditions due to accumulation of NADH. We investigated the possibility of converting this excess NADH to NAD⁺ by transforming a double mutant (gpd1∆gpd2∆) with alternative oxidoreductase genes that might restore the redox balance and produce either sorbitol or propane-1,2-diol. All of the modifications improved fermentative ability and/or growth of the double mutant strain in a self-generated anaerobic high sugar medium. However, these strain properties were not restored to the level of the parental wild-type strain. The results indicate an apparent partial NAD⁺ regeneration ability and formation of significant amounts of the commodity chemicals like sorbitol or propane-1,2-diol. The ethanol yields were maintained between 46 and 48% of the sugar mixture. Other factors apart from the maintenance of the redox balance appeared to influence the growth and production of the alternative products by the genetically manipulated strains.     

Galactose regulation in Saccharomyces cerevisiae. The enzymes encoded by the GAL7, 10, 1 cluster are co-ordinately controlled and separately translated.

The enzymes for galactose metabolism in Saccharomyces cerevisiae are encoded by three tightly linked genes. Data presented in this paper show that, in contrast to enzymes encoded by other gene clusters in yeast, these three enzymes are translated as separate polypeptides. First, two of the enzymes encoded by the cluster, galactokinase and uridylyl transferase. purified to near homogeneity, are separate polypeptides. Second, no precursor polypeptide-containing sequences common to both these enzymes is detectable in extracts from galactose-induced yeast cells. Third, no partial or absolute polarity of expression of the enzymes is observed in strains containing nonsense mutations in any of the genes of the cluster.Expression of the three galactose metabolic enzymes is co-ordinate, both during induction and during steady-state synthesis. This is true both for wild-type yeast strains and for strains carrying the long-term galactose adaptation mutation, gal3. In GAL3⁺ strains mutations within the galactose gene cluster have no effect on this co-ordinate expression. However, in gal3^? strains, mutations in any of the genes of the cluster completely eliminate expression of the other two genes. These results suggest that the GAL3 gene product is responsible for inducer synthesis and that the actual inducer is an intermediate in galactose metabolism.     

Cloning and characterization of seven cDNAs for hyperosmolarity-responsive (HOR) genes of Saccharomyces cerevisiae

Yeast cells can respond and adapt to osmotic stress. In our attempt to clarify the molecular mechanisms of cellular responses to osmotic stress, we cloned seven cDNAs for hyperosmolarity-responsive (HOR) genes from Saccharomyces cerevisiae by a differential screening method. Structural analysis of the clones revealed that those designated HOR1, HORS, HOR4, HOR5 and HOR6 encoded glycerol-3-phosphate dehydrogenase (Gpd1p), glucokinase (Glklp), hexose transporter (Hxtlp), heat-shock protein 12 (Hsp12p) and Na⁺, K⁺, Li⁺-ATPase (Enalp), respectively. HOR2 and HOR7 corresponded to novel genes. Gpdlp is a key enzyme in the synthesis of glycerol, which is a major osmoprotectant in S. cerevisiae. Cloning of HOR1/GPD1 as a HOR gene indicates that the accumulation of glycerol in yeast cells under hyperosmotic stress is, at least in part, caused by an increase in the level of GPDH protein. We performed a series of Northern blot analyses using HOR cDNAs as probes and RNAs prepared from cells grown under various conditions and from various mutant cells. The results suggested that all the HOR genes are regulated by common signal transduction pathways. However, the fact that they exhibited certain distinct responses indicated that they might also be regulated by specific pathways in addition to the common pathways. Ca²⁺ seemed to be involved in the signaling systems. In addition, Hog1p, one of the MAP kinases in yeast, appeared to be involved in the regulation of expression of HOR genes, although its function seemed to be insufficient for the overall regulation of expression of these genes.     

Performance of the auxotrophic Saccharomyces cerevisiae BY4741 as host for the production of IL-1β in aerated fed-batch reactor: role of ACA supplementation, strain viability, and maintenance energy

Background

Pichia pastoris is widely used as a production platform for heterologous proteins and model organism for organelle proliferation. Without a published genome sequence available, strain and process development relied mainly on analogies to other, well studied yeasts like Saccharomyces cerevisiae.

Results

To investigate specific features of growth and protein secretion, we have sequenced the 9.4 Mb genome of the type strain DSMZ 70382 and analyzed the secretome and the sugar transporters. The computationally predicted secretome consists of 88 ORFs. When grown on glucose, only 20 proteins were actually secreted at detectable levels. These data highlight one major feature of P. pastoris, namely the low contamination of heterologous proteins with host cell protein, when applying glucose based expression systems. Putative sugar transporters were identified and compared to those of related yeast species. The genome comprises 2 homologs to S. cerevisiae low affinity transporters and 2 to high affinity transporters of other Crabtree negative yeasts. Contrary to other yeasts, P. pastoris possesses 4 H⁺/glycerol transporters.

Conclusion

This work highlights significant advantages of using the P. pastoris system with glucose based expression and fermentation strategies. As only few proteins and no proteases are actually secreted on glucose, it becomes evident that cell lysis is the relevant cause of proteolytic degradation of secreted proteins. The endowment with hexose transporters, dominantly of the high affinity type, limits glucose uptake rates and thus overflow metabolism as observed in S. cerevisiae. The presence of 4 genes for glycerol transporters explains the high specific growth rates on this substrate and underlines the suitability of a glycerol/glucose based fermentation strategy. Furthermore, we present an open access web based genome browser http://www.pichiagenome.org.     

Insights into the Dekkera bruxellensis Genomic Landscape: Comparative Genomics Reveals Variations in Ploidy and Nutrient Utilisation Potential amongst Wine Isolates

The yeast Dekkera bruxellensis is a major contaminant of industrial fermentations, such as those used for the production of biofuel and wine, where it outlasts and, under some conditions, outcompetes the major industrial yeast Saccharomyces cerevisiae. In order to investigate the level of inter-strain variation that is present within this economically important species, the genomes of four diverse D. bruxellensis isolates were compared. While each of the four strains was shown to contain a core diploid genome, which is clearly sufficient for survival, two of the four isolates have a third haploid complement of chromosomes. The sequences of these additional haploid genomes were both highly divergent from those comprising the diploid core and divergent between the two triploid strains. Similar to examples in the Saccharomyces spp. clade, where some allotriploids have arisen on the basis of enhanced ability to survive a range of environmental conditions, it is likely these strains are products of two independent hybridisation events that may have involved multiple species or distinct sub-species of Dekkera. Interestingly these triploid strains represent the vast majority (92%) of isolates from across the Australian wine industry, suggesting that the additional set of chromosomes may confer a selective advantage in winery environments that has resulted in these hybrid strains all-but replacing their diploid counterparts in Australian winery settings. In addition to the apparent inter-specific hybridisation events, chromosomal aberrations such as strain-specific insertions and deletions and loss-of-heterozygosity by gene conversion were also commonplace. While these events are likely to have affected many phenotypes across these strains, we have been able to link a specific deletion to the inability to utilise nitrate by some strains of D. bruxellensis, a phenotype that may have direct impacts in the ability for these strains to compete with S. cerevisiae.